In vitro analysis of MPLT487A cells. (A) Growth of myeloid colonies from bone marrow cells in the presence (+) or the absence (−) of cytokines. MPLW515L- and MPLT487A-transduced, but not MPLWT-transduced, cells are able to generate myeloid colonies in the absence of cytokines. Two MPLW515L, 5 MPLT487A, and 3 MPLWT animals were analyzed. (B) Growth of myeloid colonies from spleen cellular suspensions in the presence (+) or absence (−) of cytokines. Consistent with the observed splenomegaly, MPLW515L and MPLT487A mice generate myeloid colonies in the presence or absence of cytokines. Two MPLW515L, 5 MPLT487A, and 3 MPLWT animals were analyzed. (C) Megakaryocytic progenitor colony assays. Bone marrow samples of MPLT487A mice generated more megakaryocytic colonies than bone marrow from MPLWT mice (P = .01). Two MPLW515L, 5 MPLT487A, and 4 MPLWT animals were analyzed. Error bars denote SD. (D) Acetylcholine staining of the megakaryocytic colonies. As for MPLW515L, megakaryocytic colonies from MPLT487A mice were larger than those from MPLWT mice. (E) Alignment of human (top) and mouse (bottom) MPL proteins. Amino acids 474 to 522 (NP 005364) are shown for the human MPL protein and 465 to 513 (NP 034953) for the mouse MPL. The predicted transmembrane region is boxed. Arrowheads indicate the amino acids mutated in human hematologic malignancies. Immunohistochemistry-stained sections were examined under a Leica DMLB microscope (Leica, Heidelberg, Germany) equipped with a 10× eyepiece and a 10×/0.40 CS HC PL APO objective lens. Images were captured with a Sony Power HAD camera (Sony France, Paris, France) and imaging was performed with the version 1.4 software TRIBVN-ICS (Image Communication Software, Chatillon, France) and Adobe Photoshop version 7.0 software (Adobe Systems, San Jose, CA).